The insect epidermis which produces the overlying cuticle (exoskeleton) performs its various roles in a precisely coordinated manner regulated by the hormonal milieu. Moreover, the epidermal cell in many insects undergoes a sequential polymorphism controlled by the two hormones, 20 hydroxyecdysone (20HE) and juvenile hormone (JH). Therefore, to prove more deeply into the nature and control of these processes, we propose to continue our studies on the epidermis of the tobacco hornworm, Manduca sexta, as a model system. Our specific aims are: to isolate the insecticyanin gene from the Manduca genomic clone library and use it as a representative larval gene to study the regulation of its synthesis by 20HE and JH during the molt cycle and its permanent turnoff at metamorphosis; to study the hormonal control of larval cuticular melanization and associated epidermal pigmentation with special reference to the synthesis and activities of the enzymes involved; to analyze the sequential nature of pupal cuticle deposition in vivo and in vitro, both cytologically and biochemically (in terms of changing protein patterns), the elucidate the role of ecdysteroids in its regulation utilizing the in vitro culture system; to analyze the substrates and enzymes involved in pre-ecdysial pupal tanning and the control of this process by ecdysteroids; to explore the biochemistry of the secretion of the dermal (Verson's) gland and its endocrine regulation as the basis for a possible model system for a study of the morphogenetic action of JH. The information gained in these studies can be readily applied to other insects such as vectors of disease whose epidermis is not soreadily available and utilized in devising insect control methods related to either cuticle synthesis, sclerotization, and waterproofing or to the hormonal control of these processes. Moreover, the information gained about the cellular and molecular basis of the morphogenetic action of 20HE and JH will contribute to a better understanding of a fundamental problem in developmental biology, i.e. how is a cell committed to a particular differentiative pathway.